Cancer development requires alterations in the regulatory mechanisms that control cell proliferation, apoptosis and terminal differentiation. This project is centered on the retinoblastoma protein (pRB in humans, pRb in mouse) tumor suppressor. pRB plays a key role in each of these biological processes by directly regulating their underlying transcriptional programs. Numerous pRB-responsive mRNAs have been identified and more recent studies show a direct role for pRB in the regulation of miRNAs. However, it remains unclear how the various functions of pRB contribute to its tumor suppressive roles. This project (Project 3) will investigate the mechanisms that underlie two key pRb functions. Experiments in Aims 1 and 2 will determine how mlRNA regulation contributes to pRb's role in mesenchymal differentiation and tumorigenesis. The rationale for these studies is twofold. First, we have shown that pRb is a key determinant of mesenchymal specification, and that pRb loss promotes dedifferentiation and cellular plasticity. Second, in collaboration with Project 1, we have shown that Rb family loss causes the deregulation of both well-studied (e.g. the miR-17~92 cluster) and poorly characterized miRNAs that include candidate regulators of mesenchymal plasticity. In Aim 1, we will use cell-based assays to determine the function of these pRb-regulated miRNAs including establishing the mechanism of their regulation by pRb, the identity of their targets and their roles in mesenchymal differentiation and pRb regulated plasticity. In Aim 2, we will directly test how the miR-17~92 cluster, and miRNAs more broadly, influence the growth and plasticity of tumors arising in a mouse model of Rb mutant osteosarcoma. In parallel with these mesenchymal studies, we will investigate the mechanistic basis for pRb's pro-apoptotic role. We have shown that chemotherapeutic treatment promotes formation of a transcriptionally active pRb-E2F1 complex that selectively induces apoptotic genes. Our data suggest that post-translational modifications of both E2f 1 and pRB control the formation of this complex. Experiments in Aim 3 will use gain-of-function and loss-of-function mutants to establish how these post-translational modifications affect pRB and E2f1 's ability to regulate the mRNA and miRNA programs that underlie the roles of these proteins in apoptosis versus cell proliferation.